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Structural characterization of human aryl sulphotransferases
L A Brix1, R G Duggleby, A Gaedigk
1Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland, 4072, Australia.
The Biochemical Journal
|January 12, 1999
Summary
Investigating human aryl sulphotransferases (HASTs) reveals that substrate specificity for p-nitrophenol is determined by a single amino acid change (A146E) in HAST1. Altering dopamine specificity requires coordinated changes in multiple amino acids.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Human aryl sulphotransferases (HASTs) 1, 3, 4, and 4v exhibit high sequence identity but distinct substrate specificities for dopamine and p-nitrophenol.
- Understanding the molecular basis for these differing substrate specificities is crucial for enzyme engineering and drug development.
Purpose of the Study:
- To identify the specific amino acid residues responsible for the differential substrate specificities among HAST isoforms.
- To elucidate the role of N-terminal regions and specific amino acids in HAST substrate binding and catalysis.
Main Methods:
- Construction and analysis of chimeric HAST proteins to map regions influencing substrate specificity.
- Site-directed mutagenesis of HAST1, particularly within highly divergent N-terminal Regions A and B, to mimic HAST3 residues.
- Enzyme kinetic analysis, including determination of Ki values for the co-substrate 3'-phosphoadenosine 5'-phosphosulphate (PAPS), to assess substrate binding and catalytic activity.
Main Results:
- Chimeric analysis indicated that N-terminal regions (Regions A: amino acids 44-107, and B: amino acids 132-164) largely dictate enzyme affinities.
- A single amino acid substitution (A146E) in HAST1 significantly altered its specificity for p-nitrophenol, matching that of HAST3.
- Modifying HAST1 towards HAST3 for dopamine specificity required multiple amino acid changes, with some mutations impairing co-substrate binding or activity. Deletion of residues P87-P90 in HAST1 resulted in altered substrate specificity, suggesting a role for this loop region.
Conclusions:
- A single amino acid at position 146 is a key determinant of p-nitrophenol substrate specificity in HASTs.
- Altering dopamine substrate specificity in HASTs necessitates coordinated changes in multiple amino acids within the N-terminal regions.
- Specific amino acid residues and loop regions within the N-terminus play critical roles in modulating HAST substrate binding and catalytic function.